Effect of calcium on biofilm development and role of mopB and msrA in virulence of Xylella fastidiosa

Abstract

Xylella fastidiosa is the causal agent of many diseases on economically important plants. Pierce’s disease of grape (PD) and citrus variegated chlorosis (CVC) are among the most important diseases caused by X. fastidiosa. Disease symptoms including marginal leaf scorch and shriveling of fruit are associated with the blockage of water flow in xylem vessels, caused by extensive colonization of the host xylem system by X. fastidiosa. Twitching motility and biofilm formation are considered virulence traits of this pathogen.

Research conducted in our laboratory has proven that calcium (Ca), a mineral nutrient in xylem sap, influences virulence traits of X. fastidiosa. Ca increases biofilm formation by affecting the initial stages of biofilm development. However, the effect of Ca on the later stages of biofilm development have not been characterized. In this present study, biofilm and planktonic growth over time of X. fastidiosa ‘Temecula’ cultured in different Ca-amended media was quantified. Results show that when cells are grown in non-Ca amended medium (0.02mM Ca), by 96 hours they have already reached the dispersion stage, while cells in Ca-supplemented medium (4 mM) are still forming dense, mature biofilms over the same time period. These results indicate that Ca supplementation contributes to prolong biofilm development of X. fastidiosa strain ‘Temecula’.

In addition, in order to provide information for further understanding the mechanism of X. fastidiosa response to Ca, two putative Ca-related genes mopB and msrA were selected to focus our studies. The role of these two genes in virulence traits in vitro and in disease development in vivo was evaluated. mopB and msrA mutants in two X. fastidiosa strains ‘Temecula’ and ‘WM1-1’ were constructed by site-directed mutagenesis. All knockouts had similar response to Ca supplementation as the respective WT strains, indicating that these particular genes are not responsible for the increase in biofilm and/or movement observed in WT strains after Ca supplementation. Nevertheless, mopB mutants in both background strains were impaired in surface attachment, biofilm formation, and twitching motility. In addition, mopB mutants were impaired in pilus formation as observed by electron microscopy. mopB mutants in both backgrounds showed reduced virulence when tested on tobacco as a host under greenhouse conditions. In contrast, msrA mutation in either background strain had no effect on virulence traits and disease development. These results suggest that outer membrane protein MopB is required for biofilm formation, motility, pilus biogenesis and virulence of X. fastidiosa, but msrA gene play no role in these processes under the conditions evaluated.

Overall, these results show that Ca can prolong biofilm development of X. fastidiosa, which is important to understand the disease progression in plants. MopB is important for virulence of X. fastidiosa but MsrA is not a major virulence determinant for this pathogen. Both genes are not responsible for Ca-enhanced biofilm formation and/or twitching motility in X. fastidiosa.